The present invention relates to lipoprotein particles, a process for preparing such particles and their use. In particular, the invention relates to non-naturally occurring low density lipoprotein particles, methods for their manufacture and use thereof.
Low density lipoprotein (LDL) is a natural component of plasma which is involved in the transport of cholesterol in the form of cholesterol esters around the body. Naturally occurring LDL is known to occur as roughly spherical-shaped particles (20-22 nm in diameter) which comprise an internal core of about 1500 cholesterol esters containing small amounts of triglyceride (TG). The internal core is typically surrounded by a solubilising monolayer of about 800 phospholipid molecules and small quantities of free cholesterol (about 500 molecules). Located in the monolayer is a large receptor protein, Apo protein B, of approx 500,000 daltons, (Apo B) which accounts for about twenty percent of the weight of the LDL particle.
Naturally occurring LDL finds use in a number of areas, for example, in studies on atherosclerosis and lipid metabolism. LDL also finds use as a drug-targeting vector in cancer chemotherapy. Certain cancerous cells display high rates of receptor mediated LDL uptake relative to normal cells and as such, LDL has also found use as a targeting vector for anti-cancer drugs.
Currently, naturally occurring LDL needs to be isolated from fresh plasma samples. The isolation procedure is lengthy (e.g. up to 48 hours) and depending on the donor""s plasma LDL levels the yield of LDL can be any amount up to about 100 mg ApoB/100 ml plasma in healthy individuals. Thus, yields are generally low.
Isolated naturally occurring LDL is known to be unstable. Attempts have been made to produce LDL-like particles, generally in the form of microemulsions of similar size and lipid composition to naturally occurring LDL, however such particles lack receptor competency. Apo B may be grafted onto such microemulsion particles, however, the grafting process still requires a source of the protein from fresh plasma.
Apo B is difficult to graft onto microemulsion particles partly because of its large size and a tendency for it to aggregate due to its amphipathic character. As such, grafting of Apo B onto microemulsion particles is not satisfactory because of inter alia inherent problems associated with the grafting-on process, and instability of the Apo B component.
It has now been found that a non-naturally occurring LDL can be produced which possesses LDL receptor competency, yet does not require the use of substantially whole Apo B or substantially whole analogues thereof. Furthermore, a process for the production of non-naturally occurring LDL has been developed which does not require the use of plasma derived LDL and/or plasma derived Apo B.
An object of the present invention is to provide a non-naturally occurring LDL possessing Apo B receptor competence.
Another object of the present invention is to provide a process for producing non-naturally occurring LDL particles.
These and other objects of the invention will become apparent from the following description and examples.
According to a first aspect of the present invention there is provided a non-naturally occurring, receptor competent LDL particle comprising at least one peptide component wherein the said peptide component comprises at least a binding site for an Apo B protein receptor and at least one lipophilic substituent.
A non-naturally occurring LDL particle is one which is not found occurring naturally in vivo. A non-naturally occurring LDL must be receptor competent i.e. capable of binding to Apo B receptors and/or capable of eliciting an Apo B protein-like physiological effect on and/or after binding. Thus, the non-naturally occurring LDL particle comprises at least a sequence of amino acids such as a protein, polypeptide or peptide capable of binding to Apo B receptors, which polypeptide may or may not be identical in respect of its binding region with the amino acid sequence of an Apo-B binding site, for example, an Apo B 100 binding site or physiologically functional peptide analogues thereof. Naturally, the skilled addressee will appreciate that the polypeptide capable of binding to Apo B receptors on target cells, such as cancer cells expressing Apo B receptors, is able to elicit an Apo B protein-like physiological effect on and/or after binding i.e. to be receptor competent.
The LDL particle comprises at least two components, a lipid component (L-component) and a peptide component (P-component). The L-component generally comprises a lipid emulsion comprising a core of lipophilic molecules such as cholesteryl esters, for example, cholesterol oleate, cholesterol linoleate, cholesterol stearate and the like. Other suitable lipophilic core molecules can comprise triglycerides, for example, triolein, plant oils such as soya bean oil and even lipophilic drugs, for example, estramustine, prednimustine and lipophilic modifications of known drugs, such as anti-cancer drugs, for example, cholesteryl esters of methotrexate and the like. The core of the L-component is typically solubilised by a lipid, such as an amphiphilic lipid comprising a charged or hydrophilic group. Such amphiphilic lipids include unesterified cholesterol and suitable non-ionic surfactants as well as phospholipids such as phosphatidyl choline, sphingomyelin and phosphatidyl glycerol. Preferably, the cholesteryl esters are solubilised by a monolayer of phospholipid. The preparation of the L-component is known in the art and may be performed using a variety of methods as described in the art, e.g. Ginsburg, G. S. et al (1982) J. Biol. Chem 257 (14) pp 8216-8227; Owens M. D. and Halbert G. W. (1993) J. Pharm. Pharmacol. 45 (Suppl.) p68P; Owens M. D. and Halbert G. W. (1995) Eur. J. Pharm. Biopharm 41 (2) pp 120-126, herein incorporated by reference.
Preferably, the L-component is made up of at least two biologically acceptable components. A first component can be a biologically acceptable saturated or unsaturated long chain charged polar component such as a phospholipid. Examples of suitable charged polar components include phosphatidyl choline (PC), phosphatidyl serine (PS), phosphatidyl glycerol (PG), sphingomyelin, and unesterified cholesterol and the like. The second component can be a biologically acceptable lipophilic component such as a cholesteryl ester, for example cholesteryl oleate. Biologically acceptable components are ones which may be administered to cells in vitro or in vivo and which have substantially no deleterious effect on cell viability. In a preferment the L-component can comprise three or more components in a defined ratio, such as a molar ratio, for example, phospholipid; triolein; cholesteryl ester (P:T:C) The molar ratio may be in any molar ratio as long as the components are capable of forming an L-component suitable for use in the preparation of non-naturally occurring LDL particles of the present invention. The molar ratio of outer core solubilising lipid such as phospholipid (PL) sphingomyelin (SM), phosphatidyl choline (PC) and unesterified cholesterol (UC) to core lipid such as cholesteryl ester (CE), triolein (TO) cholesteryl oleate (CO) or lipophilic drug can be in the range of from about 0.7:1 up to 5:1, preferably 1:1 to 3:1 depending on design. A preferred ratio of PL:CE is about 2:1. Where a third L-component is not employed the ratio of PL:CE can be in the range of from about 1:1 to about 2:1. A suitable molar ratio for a three component system such as a phosphatidylcholine: triolein: cholesteryl oleate is 3:2:1 respectively.
A suitable molar ratio for a five component system comprising three outer core lipids and two core lipids may lie in the range of from 0.7-6.5:0-2:0-1 (outer core lipid): 0-5:0-2.5 (core lipid). Preferably, the molar ratio lies in the range of from 2.5-4.5:1-2:0.5-1 (outer core lipid): 2-4.5:1-2.5 (core lipid). More preferably the molar ratio lies in the range of from 4-4.5:1.5-2:0.7-0.9 (outer core lipid): 4-4.5:1.8-2.2 (core lipid). Suitable outer core lipids may be selected from PC, SM, UC and PL. Suitable core lipids may be selected from TO, CE and CO. The man skilled in the art will appreciate that other suitable outer core lipids and core lipids may be used in the present invention. An example of a five component system is PC:SM:UC (outer core lipid): TO:CO (core lipid). The components of such a five component system may be present in molar ratios as indicated above.
Generally, the droplet diameter of lipid microemulsions employed in the non-naturally occurring lipoprotein particles of the invention should be capable of functioning as lipoprotein particles in vivo, ex vivo or in vitro. The diameter of the non-naturally occurring LDL particles can be up to about 50 nm, preferably from about 10 nm up to about 35 nm depending on the method of preparation and/or molar ratio such as a PL:CE molar ratio, employed.
Peptide components for use in forming LDL particles a: the invention contain at least one lipophilic substituent or moiety capable of acting as an xe2x80x9canchorxe2x80x9d for anchoring the peptides to the L-component. Lipophilic moieties or substituents may be derived from biologically compatible lipophilic compounds such as cholesterol, retinoic acid, C10-C22 fatty acids such as stearic acid (C18) and the like. The lipophilic moiety/substituent can be placed in contact with the amino and/or carboxy terminus of the peptide via chemical means such as covalent bonding or ionic bonding known in the art. The man skilled in the art will appreciate that peptides of the invention can be assembled using standard Fmoc protocols of the Merrifield solid phase synthesis method. The lipophilic substituent, such as retinoic acid can be activated and attached to the peptide N-terminus using a standard peptide coupling cycle. Initially an acid labile linker such as 3-methoxy-4-hydroxymethylphenoxyacetic acid is attached to the resin support and esterified with the first amino acid (C-terminus) of the target peptide. When peptide assembly is complete the ester to the linker can be hydrolysed, allowing removal of the fully protected peptide, for example with trifluoroacetic acid (TFA) eg. 1% TFA, in dichloromethane which can subsequently be evaporated off. At such a stage, the available functional group is the peptide carboxyl, which can be activated with for example one equivalent of dicyclohexylcarbodiimide (DCC) in dimethylformamide (DMF) and coupled to a lipophilic molecule, such as cholesterol (10 equiv), to yield ester. Evaporation of the solvent and treatment with TFA, e.g. 95% TFA, deprotects the amino acid side chains, completing the synthesis. The complete peptide can then be concentrated and precipitated with, for example, diethyl ether to give a solid which can then be washed as necessary to remove any remaining protecting group fragments and excess cholesterol.
N-terminal modifications, such as retinoic acid and stearate addition, targeted at primary amines can be used in the synthesis of modified peptides of the invention using techniques known in the art. Preferably, peptides capable of being utilised in the invention are amphipathic in nature, i.e. possess lipophilic and hydrophilic groups. Suitable hydrophilic groups include hydroxyl, carboxylic and amino groups. Where the peptides are amphipathic in character, the hydrophobic group and hydrophilic groups may be located at any suitable point thereon via appropriate side chains. Preferably the hydrophobic groups and hydrophilic groups are located either at the amino terminus and carboxy terminus of the peptide respectively or vice versa.
In an aspect of the invention there is provided non-naturally occurring LDL particles comprising peptides wherein the amino acid sequence of the binding site of said peptides are selected from the group: amino acids having basic side chains, amino acids having aliphatic side chains, and amino acids having aliphatic hydroxyl side chains.
In this aspect of the invention, the peptides of the non-naturally occurring LDL of the invention may display substantial, little or no similarity and/or identity with the amino acid sequence of the Apo B binding region. Preferably, the peptides display substantial similarity and/or identity with the amino acid sequence of the Apo B binding region.
The amino acid sequence which makes up the peptide capable of being grafted onto the lipid component of the LDL of the present invention can be selected from the group of amino acids having basic side chains e.g. lysine, arginine and histidine; amino acids having aliphatic side chains e.g. glycine, alanine, valine, leucine and isoleucine; amino acids having aliphatic hydroxyl side chains e.g. serine and threonine, and derivatives thereof.
Where the binding region amino acid sequence is substantially dissimilar to the binding region sequence of Apo B with respect to the order of amino acids incorporated thereinto, the amino acids selected for inclusion into the binding region of the amino acid sequence can be selected from substantially the same amino acids as those making up the Apo B binding region sequence. Naturally, the skilled addressee will understand that conservative replacement and/or substitutions as herein described may also be made to such binding regions.
In a preferment there is provided a non-naturally occurring LDL particle comprising at least one amino acid sequence including an Apo B binding region capable or interacting with an Apo B receptor.
Naturally, the skilled addressee will appreciate that such amino acid sequences making up functional peptides or polypeptides suitable for use in the present invention must be receptor competent as defined herein. Thus, synthetic or semi-synthetic peptides and/or polypeptides and analogues thereof capable of binding to Apo B receptors are encompassed by the present invention.
In a preferment, the amino acid sequence can comprise either or both of the Apo B binding site sequence(s) depicted below in the same peptide or in the form of dimers or in different peptides:
(1) Lys Ala Glu Tyr Lys Lys Asn Lys His Arg His (SEQ ID NO: 1);
or
(2) Thr Thr Arg Leu Thr Arg Lys Arg Gly Leu Lys (SEQ ID NO: 2);
and analogues thereof which are capable of binding to the Apo B100 receptor site.
The amino acid sequence can be of any length provided that it is capable of being grafted onto the lipid component under grafting conditions as described herein. The amino acid sequence may include sequences of up to about 500 amino acid residues long comprising sequences (1) and/or (2) above. Sequences (1) and (2) are known Apo B binding site sequences identified from the human Apo-100 protein as described by Knott T. J. et al Nature Vol. 323 October 1986 p 735. For example, an amino acid sequence could comprise the sequence from amino acid 3079 to about position 3380 of FIG. 1, p 735 (Knott et al supra). The amino acid sequence can comprise at least a single Apo B binding site sequence and can be from about 8-200 amino acid residues in length, or a shorter sequence of from about 8-50 amino acid residues in length, preferably from about 9 to 30 amino acid residues in length. Examples of suitable peptide sequences include those as depicted in FIG. 7 herein. Naturally, the skilled addressee will appreciate that practical considerations such as the ability of the amino acid sequence to bind to receptor and ability to synthesise the peptide sequence generally means that the shorter amino acid sequences are preferred. The skilled addressee will appreciate that natural variations in the amino acid sequences comprising amino acid substitutions, deletions and/or replacements are encompassed by the present invention. Furthermore, the skilled addressee will also appreciate that amino acid substitutions, deletions and/or replacements can be made to the amino acid sequence so long as such modifications do not substantially interfere with the ability of the amino acid sequence to bind to a binding site and thereby elicit a physiological response. For example, conservative replacements may be made between amino acids within the following groups:
(i) Lysine and arginine;
(ii) Alanine, serine and threonine;
(iii) Glutamine and asparagine;
(iv) Tyrosine, phenylalanine and tryptophan; and
(v) Leucine, isoleucine, valine and methionine.
so long as the physiological function of the peptide is not substantially impaired.
The invention further includes substantially isolated proteins having a substantially similar activity/function to the peptides of the instant invention and which have an amino acid sequence which comprises at least 70% similarity, that is, identity, to the sequence of peptide (1) and/or peptide (2) above when aligned optimally therewith. It is preferred that such a degree of similarity, that is identity, is at least 80%, more preferred that such a degree of similarity is 90% or higher. In the context of the present invention two amino acid sequences with at least 70% similarity to each other have at least 70% identical or conservatively replaced amino acid residues in a like position when aligned optimally using computer programs known in the art, such as BLAST and FASTA.
In a further aspect of the invention there is provided use of non-naturally occurring LDL particles as drug-targeting vectors. Such drug targeting vectors can be used in the treatment of cancerous cells and the like.
In another aspect of the invention there is provided a process of producing a non-naturally occurring, receptor competent LDL particle possessing Apo B receptor competency which comprises:
i) forming a lipid component from lipophilic molecules and amphiphilic lipid molecules; and
ii) contacting at least one peptide with a lipid component formed in i) wherein the at least one peptide comprises at least a lipophilic substituent and at least a binding region capable of interaction with an apoprotein B receptor.
In a further aspect of the invention there is provided use of an nLDL particle as a drug targeting vector, in particular in the treatment of cancerous cells comprising Apo B protein receptors in a patient.
In a still further aspect of the invention there is provided use of an nLDL particle in the manufacture of a medicament for the treatment of disease, in particular cancer. Thus, there is provided use of an nLDL particle in the manufacture of a drug targeting vector comprising a medicament therein for the treatment of cancerous cells comprising Apo B protein receptors.
The skilled addressee will appreciate that non-naturally occurring, receptor competent LDL particles of the invention may be administered to a patient along with conventional vehicles or carriers typically used in drug and vesicle presentation such as water, physiologically acceptable saline solutions, buffers and the like.
As a further aspect of the invention there is provided a Pharmaceutical formulation comprising a non-naturally occurring receptor competent LDL together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may be selected from conventional carriers commonly employed in the art.
Additionally, there is provided a method for the treatment of cancer in a patient which comprises administering to the patient a clinically useful amount of a non-naturally occurring LDL particle comprising an anti-cancer drug.
The lipid component is as hereinbefore described as are the types of peptides which may be employed.